246 J Med Genet 1999;36:246–250

Skin pigmentary anomalies and mosaicism for an J Med Genet: first published as 10.1136/jmg.36.3.246 on 1 March 1999. Downloaded from acentric marker chromosome originating from 3q

Marie-France Portnoï, Serge Boutchneï, Fabrice Bouscarat, Geneviève Morlier, Sonia Nizard, Hera Dersarkissian, Béatrice Crickx, Marc Nouchy, Jean-Louis Taillemite, Stéphane Belaich

Abstract The lines of Blaschko are linear areas of skin We report on a 22 year old man with diVerentiated from adjoining skin only by a hyperpigmentation distributed along the diVerence in pigmentation. They are aligned Service d’Embryologie lines of Blaschko in whom cytogenetic along the axis of limbs and circumferentially Pathologique et de around the trunk, and are thought to represent Cytogénétique, Hôpital analysis showed mosaicism for an unusual Saint-Antoine AP-HP, supernumerary marker chromosome. the tracts of migration of melanocytes. The 75012 Paris, France The patient was of normal intelligence and pattern outlined by Blaschko lines shows M-F Portnoï was not dysmorphic. The marker was genetic mosaicism that has arisen in the G Morlier present in 30% of his lymphocytes and in embryo before the development of neural folds M Nouchy and reflects the dorsoventral outgrowth of two 6% of his skin fibroblasts from a dark area, J-L Taillemite genetically diVerent populations of cells.1 while fibroblasts from a light area showed Numerous and diverse chromosomal abnor- Service de a normal karyotype, 46,XY.We have iden- Dermatologie, Hôpital malities have been reported in patients with d’Instruction des tified the origin of the marker using pigmentary anomalies distributed along the Armées R Picqué, fluorescence in situ hybridisation (FISH) lines of Blaschko, characterised by the presence 33998 Bordeaux with whole chromosome painting probes of mosaicism in peripheral blood lymphocytes Armées, France and YAC specific clones. The marker was 2–5 S Boutchneï or skin fibroblast cultures. Many patients found to consist of duplicated chromo- have, in addition, various patterns of multiple Service de some material from the distal part of congenital anomalies and mental retardation. Dermatologie, Hôpital chromosome 3q and was interpreted Chromosomal aberrations may result in abnor- Bichat AP-HP,75018 as inv dup(3)(qter→q27.1::q27.1→qter). malities of pigmentation because of the altered Paris, France Hence, this marker did not include any expression of genes in the pigmentary pathway, F Bouscarat known centromeric region and no alpha which map to a number of diVerent B Crickx 67 S Belaich satellite DNA could be detected at the site chromosomes. of the primary constriction. The patient We report here on a patient who displayed

CEPH-Hôpital was therefore tetrasomic for 3q27-q29 in skin pigmentary anomalies distributed along http://jmg.bmj.com/ Saint-Louis AP-HP, the cells containing the marker chromo- the lines of Blaschko and was mosaic for a small 75010 Paris, France supernumerary marker chromosome. The in- S Nizard some. We postulate that, in our case, H Dersarkissian pigmentary anomalies may result directly troduction of molecular cytogenetic techniques from the gain of specific pigmentation has greatly increased the possibility for full Correspondence to: genes localised on chromosome 3q. characterisation of marker chromosomes. Fluo- Dr Portnoï. (J Med Genet 1999;36:246–250) rescence in situ hybridisation (FISH) tech- niques show that markers can be derived from a Received 26 March 1998 89 Revised version accepted for Keywords: pigmentary anomalies; acentric marker variety of chromosomes. Using FISH, the on September 24, 2021 by guest. Protected copyright. publication 14 July 1998 chromosome; mosaicism; tetrasomy 3q results of the present study showed that the marker originated from chromosome 3 and was an unusual acentric marker chromosome lack- ing detectable alpha satellite DNA. Alpha satellite DNA is believed to be important for centromeric function, because of its presence at all human centromeres.10

Materials and methods CYTOGENETIC ANALYSIS Chromosome analysis was performed on phy- tohaemagglutinin stimulated peripheral lym- phocyte and skin fibroblast cultures after GTG, RHG, and C banding using standard procedures. Skin biopsy was obtained from both light and dark areas. An EBV transformed lymphoid cell line was established.

FLUORESCENCE IN SITU HYBRIDISATION Fluorescence in situ hybridisation (FISH) was carried out on mitotic preparations according to Pinkel et al11 with whole chromosome paint- Figure 1 Photograph of the patient (reproduced with permission). ing probes (CAMBIO) and alpha satellite Skin pigmentary anomalies and chromosome 3q 247

probes either for all human centromeres the pigmentary anomalies was recorded at the (ONCOR) or specific for chromosome 3 age of 10-12 years. This was not preceded by J Med Genet: first published as 10.1136/jmg.36.3.246 on 1 March 1999. Downloaded from (D3Z1-ONCOR). Additional FISH studies inflammation or vesicles. First both legs were were performed with 30 yeast artificial chro- aVected, then the whole body, except for the mosome (YAC) clones positive for microsatel- palms, soles, eyes, and mucous membranes. lite markers known to map to chromosome 3.12 Slight itching was present. Physical examina- All YACs were supplied by the CEPH library. tion was otherwise normal. The pigmentary A subsequent FISH analysis was carried out anomalies were characterised by bilateral, with three selected YACs spanning the marker asymmetrical, linear, parallel, and hyperpig- breakpoint region (883F3, 781F8, 753C2). All mented brown macular streaks, following the probes were labelled with biotin and detected lines of Blaschko (fig 1). There was no cutane- with avidin FITC and a PI counterstain. ous atrophy, but hair growth was decreased on aVected areas. A brown spiral colouration was Case report visible on his occiput. A 22 year old man with no relevant personal Results of chest x rays, ophthalmological history and of normal intelligence was referred examination, heart and abdominal echography, because of pigmentary cutaneous anomalies. and routine biological investigations were nor- The patient was healthy and not dysmorphic. mal. Skin biopsy showed a diVuse basal layer His parents were unrelated and no pigmentary hyperpigmentation. disorder was present in his family. The onset of Results Cytogenetic analysis showed two populations of cells: a normal male cell line and a line con- taining 47 chromosomes with an extra super- numerary marker chromosome (fig 2). The marker was present in 30% of the lymphocyte metaphases and in 6% of the fibroblasts from hyperpigmented skin. It was absent in fibro- blasts from the normally coloured skin which showed a normal 46,XY karyotype in 200 mitoses analysed. The marker of the G group size was acrocentric without satellites and had no visible C band. The banding pattern was non-specific and could not be attributed to any defined chromosome region. The marker was tested by FISH with many chromosome specific libraries. FISH with a chromosome 3

specific library painted the marker chromo- http://jmg.bmj.com/ some entirely, as well as the two normal chromosomes 3, indicating that the marker originated from chromosome 3 (fig 2). The chromosome 3 alpha satellite probe did not hybridise with the marker but did hybridise to the normal chromosomes 3. An all human centromeres probe hybridised to all chromo-

somes except the marker, indicating the on September 24, 2021 by guest. Protected copyright. absence of alpha satellite sequences on the marker (fig 2). Further characterisation was achieved using YAC specific probes from chromosome 3. The marker was not labelled with the probes local- ised to 3p, but showed positive labelling with the probes (883D12, 760F3, 770A8) mapped to the distal 3q region from 3q27 to 3q29. The last negative YAC (806D8) was mapped to 3q26.3. The breakpoint was found to be local- ised in 3q27.1 using three selected YACs (883F3, 781F8, 753C2). Each positive probe produced double and symmetrical signals on the marker chromosome (fig 2). The hybrid- isation pattern suggested that the marker chromosome was an inverted duplication of the distal 3q region, interpreted as Figure 2 Chromosomes of the patient and FISH analysis of the marker chromosome. (A) 3qter→q27.1::q27.1→3qter. The two 3q27.1 G banded chromosomes; arrow indicates an additional marker chromosome. (B) bands had coalesced, becoming a large posi- Hybridisation of the chromosome 3 painting probe to the normal 3 and marker chromosomes (arrows). (C) FISH with the all chromosome centromeres probe. All tive band in the middle of the marker with the chromosomes showed signal except the marker (arrow). (D) Partial metaphase following duplicated 3q29 bands lying in the two most FISH with YAC from the 3q27 region. The two 3q27 bands had coalesced (arrow). (E) distal regions of the marker. The patient’s Partial metaphase following hybridisation with YAC probes from the 3q28 region. Arrows point to double signals on the marker and a single signal on the normal 3. (F) FISH with karyotype was: 46,XY/47,XY,+inv dup(3) YAC probes from the 3q29 region. (qter→q27.1::q27.1→qter). He was thus 248 Portnoï, Boutchneï, Bouscarat, et al

mosaic for pure tetrasomy 3q, which, to our ing Blaschko’s lines from other pigmentary knowledge, has never been described before. disorders. J Med Genet: first published as 10.1136/jmg.36.3.246 on 1 March 1999. Downloaded from The parents were not available for testing. The clinical findings of chromosomal mosai- cism associated with dermatoses are diverse, most often including dysmorphic features, Discussion various patterns of multiple congenital anoma- Pigmentary anomalies have their origin all lies, mental retardation, asymmetrical and along the path which leads from melanoblast abnormal growth patterns, and skin pigmen- production to the melanocytes and their tary anomalies; patients present with streaks of dysfunctions. The pattern of distribution in hyperpigmented skin or linear hypopigmen- cutaneous dyspigmentation may vary, but often tation described as hypomelanosis of Ito.17 follows the lines of Blaschko, as in our patient. However, there have not been any consistent The lines of Blaschko consist of transverse chromosomal findings. Similar pigmentary bands on the trunk, following a sigmoid curve dysplasias may be associated with diVerent over the abdomen, a V shaped curve over the chromosomal anomalies involving autosomes back, and showing a marked midline eVect. On or sex chromosomes, and diVerent pigmentary the limbs they follow a perpendicular pattern disorders may be associated with identical and on the scalp they form a spiral. It is now chromosomal anomalies. In their series, Ohashi well known that Blaschko’s lines can be et al4 described two patients mosaic for trisomy explained on the basis of cellular mosaicism, 18; one had hypopigmented lesions, the other chromosomal mosaicism, or mosaic distribu- hypo- and hyperpigmented skin anomalies. tion of single gene mutation.13 The pattern is Thomas et al3 reported seven patients; only one caused by the random arrangement of melano- was of normal intelligence and he had been blast precursors along the length of the neural assessed because of first degree hypospadias crest and by their relative migration potential. and linear hypopigmented lesions and lym- This particular shape is the result of the type of phocytes and skin fibroblasts showed 45,X/ embryonic growth of cutaneous tissue. Two 46,XY mosaicism.These reports indicate that clonal genetically diVerent populations of pre- multiple genes are involved in pigmentation cursor cells begin to proliferate in a transvere and map to a number of diVerent chromo- direction, starting from the primitive streak somes. each side of the neural tube. The transverse The patient described in this report was clonal proliferation of cells interferes with the mosaic for an extra supernumerary marker longitudinal growth and increasing flexion of chromosome in lymphocytes and in fibroblasts the embryo.These complex movements result from hyperpigmented skin; his karyotype from in the bizarre pattern of the lines of Blaschko.1 light skin was totally normal. We only exam- Our patient had a late, stable dyspigmenta- ined lymphocytes and fibroblasts cytogeneti- tion following Blaschko’s lines. DiVerent skin cally as it was not feasible to study cultured

pigmentary dysplasia distributed in a linear keratinocytes or melanocytes. Melanocytes are http://jmg.bmj.com/ pattern could be ruled out as follows. Inconti- not easy to grow and the patient was not avail- nentia pigmenti (IP), whose vesiculobullous able for further biopsies.Therefore, we are not first stage is sometimes absent but never late in absolutely sure that the melanocytes of the appearing, is an X linked dominant gene defect hyperpigmented or normal areas show the with lethality in hemizygous males. The marker chromosome. “acquired pigmented atrophic band-like der- The marker was identified by FISH using matose following Blaschko’s lines” of Moulin et panels of whole chromosome painting probes 14 al could be ruled out because there was no as originating from chromosome 3. Markers on September 24, 2021 by guest. Protected copyright. atrophic lesion in our patient, as could the derived from chromosome 3, identified by an “familial progressive hyperpigmentation” of alpha satellite probe specific for chromosome Chernosky et al,15 aVecting one mother and 3, have been reported in four cases.18–20 They three of her children born from two diVerent were much smaller than in our case. In one of partners, as lesions were present at birth and two cases of Muller-Navia et al,20 further char- rapidly spread in the first two years of life. The acterisation showed that the marker derived closest skin disease previously described is the from the juxtacentromeric region of 3p. Skin “linear and whorled nevoid hypermelanose” of pigmentary anomalies were not described in Kalter et al,16 consisting of 1 to 5 mm coloured, any of these four reports. The first association homogeneous macules producing a reticulated of a skin disorder with a chromosomal form with asymmetrical streaks and whorls. abnormality in chromosome 3 was reported by Lesions appear during the first weeks of life, Jokiaho et al.21 They described a 3q27-qter sometimes later, aVecting boys and girls. There deletion in a girl with meningocele, facial dys- is no previous inflammation or papular lesions. morphism, and skin lesions following the lines Lesions spread moderately during the first two of Blaschko. Happle22 suggested that the erup- years then stabilise and mucous and palmo- tion observed in this girl appeared to be a new plantar areas are spared. Congenital anomalies skin disease related to the extremely uncom- are often present. Histological examination mon deletion 3q27-qter. The similar 3q shows a non-specific increased pigmentation of chromosomal segment was involved in our the basal layer. Cytogenetic analysis of cultured marker chromosome. fibroblasts from light and dark skin of patients The marker clearly has a functional centro- was normal. Patients with skin pigmentation mere that is C band negative and forms a disorders are not described precisely enough distinct primary constriction. FISH using an clearly to diVerentiate dyspigmentation follow- alpha satellite probe either for all human Skin pigmentary anomalies and chromosome 3q 249

chromosomes or specific for chromosome 3 duces a distinct and recognisable phenotype.32 failed to detect a signal on the marker, which It resembles the phenotype in the Cornelia de J Med Genet: first published as 10.1136/jmg.36.3.246 on 1 March 1999. Downloaded from was similar to an acentric marker chromosome. Lange syndrome and includes hirsutism, syno- Occasional markers devoid of alphoid se- phrys, seizures, severe mental retardation, and quences detectable by FISH have been slow growth. Duplications may be of variable reported.18 19 23–25 It was suggested that these length, usually within the region 3q21-3qter. marker chromosomes had truncated alpha sat- Ireland et al33 proposed that 3q26.3 is the 3q ellite regions which may be impossible to visu- duplication critical region and that the de alise with FISH. Voullaire et al26 and Maraschio Lange gene maps in this area. Van Essen et al,34 et al27 have both reported a novel mechanism reviewing published reports, suggested that tri- for the formation of acentric marker chromo- somy of genes located in the distal part of 3q26 somes, derived from chromosome 1026 and and the proximal part of 3q27 are essential for from chromosome 3.27 Markers were associ- the characteristic phenotype in partial trisomy ated with a ring chromosome and formed by 3q syndrome. Our patient had mosaic tetra- the joining of the acentric portions, distal to the somy of a 3q segment distal to the critical centric region, forming the ring. Activation of a region of the duplication 3q syndrome, ex- latent intercalary centromere was suggested as plaining why none of the features associated the mechanism of formation. In our case and in with this syndrome was observed. However, it that of Maraschio et al27 the supernumerary was surprising that mosaic tetrasomy 3q27- chromosome derived from chromosome 3, but qter had no major impact on the phenotype, with a primary constriction localised in a apart from skin pigmentary anomalies distrib- diVerent region. Further investigations of the uted along the lines of Blaschko. Karyotype marker derived from chromosome 10 have analysis from biopsies of light and dark areas of been performed at a molecular level by du Sart the skin showed that the light skin contained et al.28 They provided evidence in support of only normal cells, but there was a positive cor- the formation of the marker centromere relation between the abnormal karyotype from through the activation of a latent centromere dark skin and the pigmentary abnormalities. that exists within the normal human genome. The cytogenetic abnormality may be directly The new centromere is composed of non- responsible for the linear dyspigmentation. Our satellite DNA, suggesting that the alpha observation lends support to the suggestion satellite DNA is not mandatory for centromere that this 3q27-qter region interferes with regu- function. Hence, an ancient centromere se- lation of pigmentation and is probably the quence might exist at distal 3q and was location of a gene(s) responsible for skin activated through the chromosome rearrange- pigmentation. In addition, skin lesions in a girl ment in our patient. with a deletion 3q27-qter were reported by In our case the marker chromosome was Jokiaho et al.21 identified as an inverted duplication, derived Although numerous observations of super- → →

from 3q, interpreted as 3qter q27.1::q27.1 numerary chromosomes with various cyto- http://jmg.bmj.com/ qter, without alpha satellite DNA. These genetic morphologies have been published, uncommon types of marker have recently been their clinical significance in individual cases has reported. They were found to be inversely been a major problem. In a large multicentre duplicated segments from the distal region of study 1/2500 de novo small marker chromo- 8p29 and from distal 15q30 without the centro- somes were detected among 377 353 amnio- meric region. The authors concluded that centeses during a 10 year period and abnormal these findings strongly indicate that intercalary phenotypic outcome was seen in 13% of 35 ancient centromere sequences are present and cases. There is a correlation between mental on September 24, 2021 by guest. Protected copyright. could be activated through chromosome rear- retardation, the size, its euchromatic content, rangements. These kinds of marker can be and the mutant origin of a marker.36 In our generated by a single “U type” exchange event case, the patient was intellectually normal. The involving chromosome breakage and subse- absence of severe phenotypic consequences is quent reunion between the sister chromatids. A most likely because of the mosaicism and the U type exchange can also be generated by a marker could be restricted to moderately simple abnormal event in DNA replication, as aVected tissues/organs. Therefore, our case suggested by Wik Sjostedt et al.31 These confirms the relationship between chromo- theories have been postulated concerning the somal mosaicism and anomalous skin pigmen- formation of dicentric isochromosomes and tation, particularly when it follows the lines of include the formation of an acentric fragment Blascko, and stresses the importance in these which, because of the lack of a functional cen- cases of careful evaluation, which should tromere, is assumed to be lost. It could be include a lymphocyte karyotype and karyo- speculated that this acentric fragment might types from skin biopsies from dark and light somehow be retained, if it acquired or activated skin. sequences that would function as a new 30 centromere. The formation of this type of The first two authors contributed equally to this work. We are marker chromosome may have been over- grateful to V Bruna, I Pinson, and F Rajhonson for their expert looked in the past, because of diYculties in technical assistance. We also thank S Jan for the photographs. resolving the rearrangements. 1 Happle R. Lyonization and the lines of Blaschko. Hum Genet The patient described here was thus mosaic 1985;70:200-6. for pure tetrasomy 3q, which to our knowledge 2 Turleau C, Taillard F, Doussau de Bazignan M, Delepine N, has never been described before. However, tri- Desbois JC, de Grouchy JC. Hypomelanosis of Ito and mosaicism for a microdeletion of 15q1. Hum Genet somy for distal 3q has been reported, and pro- 1986;74:185-7. 250 Portnoï, Boutchneï, Bouscarat, et al

3 Thomas JT, Frias JL, Cantu ES, Lafer C, Flannery DB, 21 Jokiaho I, Salo A, Niemi KM, Blomstedt GC, Pihkala J.

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application of microdissection in prenatal diagnosis. Hum probands with an additional “marker” chromosome. Hum http://jmg.bmj.com/ Genet 1995;96:661-7. Genet 1985;69:353-70. on September 24, 2021 by guest. Protected copyright.